IKK-β proteins, nucleic acids and methods

ABSTRACT

The invention provides methods and compositions relating to an IκB kinase, IKK-β, and related nucleic acids. The polypeptides may be produced recombinantly from transformed host cells from the disclosed IKK-β encoding nucleic acids or purified from human cells. The invention provides isolated IKK-β hybridization probes and primers capable of specifically hybridizing with the disclosed IKK-β genes, IKK-β-specific binding agents such as specific antibodies, and methods of making and using the subject compositions in diagnosis, therapy and in the biopharmaceutical industry.

This a continuing application under 35USC120 of U.S. Ser. No. 08/887,114 filed Jul. 1, 1997, abandoned.

FIELD OF THE INVENTION

The field of this invention is proteins involved in transcription factor activation.

BACKGROUND

Cytokines trigger changes in gene expression by modifying the activity of otherwise latent transcription factors (Hill and Treisman, 1995). Nuclear factor κB (NF-κB) is a prominent example of how such an external stimulus is converted into an active transcription factor (Verma et al., 1995). The NF-κB system is composed of homo- and heterodimers of members of the Rel family of related transcription factors that control the expression of numerous immune and inflammatory response genes as well as important viral genes (Lenardo and Baltimore, 1989; Baeuerle and Henkel, 1994). The activity of NF-κB transcription factors is regulated by their subcellular localization (Verma et al., 1995). In most cell types, NF-κB is present as a heterodimer comprising of a 50 kDa and a 65 kDa subunit. This heterodimer is sequestered in the cytoplasm in association with IκBα a member of the IκB family of inhibitory proteins (Finco and Baldwin, 1995; Thanos and Maniatis, 1995; Verma et al., 1995). IκBα masks the nuclear localization signal of NF-κB and thereby prevents NF-κB nuclear translocation. Conversion of NF-κB into an active transcription factor that translocates into the nucleus and binds to cognate DNA sequences requires the phosphorylation and subsequent ubiquitin-dependent degradation of IκBα in the 26s proteasome. Signal-induced phosphorylation of IκBα occurs at serines 32 and 36. Mutation of one or both of these serines renders IκBα resistant to ubiquitination and proteolytic degradation (Chen et al., 1995).

The pleiotropic cytokines tumor necrosis factor (TNF) and interleukin-1 (IL-1) are among the physiological inducers of IκB phosphorylation and subsequent NF-κB activation (Osborn et al., 1989; Beg et al., 1993). Although TNF and IL-1 initiate signaling cascades leading to NF-κB activation via distinct families of cell-surface receptors (Smith et al., 1994; Dinarello, 1996), both pathways utilize members of the TNF receptor-associated factor (TRAF) family of adaptor proteins as signal transducers (Rothe et al., 1995; Hsu et al., 1996; Cao et al., 1996b). TRAF proteins were originally found to associate directly with the cytoplasmic domains of several members of the TNF receptor family including the 75 kDa TNF receptor (TNFR2), CD40, CD30, and the lymphotoxin-β receptor (Rothe et al., 1994; Hu et al., 1994; Cheng et al., 1995; Mosialos et al., 1995; Song and Donner, 1995; Sato et al., 1995; Lee et al., 1996; Gedrich et al., 1996; Ansieau et al., 1996). In addition, TRAP proteins are recruited indirectly to the 55 kDa TNF receptor (TNFR1) by the adaptor protein TRADD (Hsu et al., 1996). Activation of NF-κB by TNF requires TRAF2 (Rothe et al., 1995; Hsu et al., 1996). TRAF5 has also been implicated in NF-κB activation by members of the TNF receptor family (Nakano et al., 1996). In contrast, TRAF6 participates in NF-κB activation by IL-1 (Cao et al., 1996b). Upon IL-1 treatment, TRAF6 associates with IRAK, a serine-threonine kinase that binds to the IL-1 receptor complex (Cao et al., 1996a).

The NF-κB-inducing kinase (NIK) is a member of the MAP kinase kinase kinase (MAP3K) family that was identified as a TRAF2-interacting protein (Malinin et al., 1997). NIK activates NF-κB when overexpressed, and kinase-inactive mutants of NIK comprising its TRAF2-interacting C-terminal domain (NIK.sub.(624-947)) or lacking two crucial lysine residues in its kinase domain (NIK_(KK429-430AA))) behave as dominant-negative inhibitors that suppress TNF-, IL-1-, and TRAF2-induced NF-κB activation (Malinin et al., 1997). Recently, NIK was found to associate with additional members of the TRAP family, including TRAF5 and TRAF6. Catalytically inactive mutants of NIK also inhibited TRAF5- and TRAF6-induced NF-κB activation, thus providing a unifying concept for NIK as a common mediator in the NF-κB signaling cascades triggered by TNF and IL-1 downstream of TRAFs.

Here, we disclose a novel kinase IκB Kinase, IKK-β, as a NIK-interacting protein. IKK-β has sequence similarity to the conceptual translate of a previously identified open reading frame postulated to encode a serine-threonine kinase of unknown function (`Conserved Helix-loop-helix Ubiquitous Kinase` or CHUK, Connelly and Marcu, 1995; Mock et al., 1995). Catalytically inactive mutants of IKK-β are shown to suppress NF-κB activation induced by TNF and IL-1 stimulation as well as by TRAF and NIK overexpression; transiently expressed IKK-β is shown to associate with the endogenous IκBα complex; and IKK-β is shown to phosphorylate IκBα on serines 32 and 36. As used herein, Ser32 and Ser36 of IκB refers collectively to the two serine residues which are part of the consensus sequence DSGL/IXSM/L (e.g. ser 32 and 36 in IκBα, ser 19 and 23 in IκBβ, and ser 157 and 161, or 18 and 22, depending on the usage of methionines, in IκBε, respectively.

SUMMARY OF THE INVENTION

The invention provides methods and compositions relating to isolated IKK-β polypeptides, related nucleic acids, polypeptide domains thereof having IKK-β-specific structure and activity and modulators of IKK-β function, particularly IκB kinase activity. IKK-β polypeptides can regulate NFκB activation and hence provide important regulators of cell function. The polypeptides may be produced recombinantly from transformed host cells from the subject IKK-β polypeptide encoding nucleic acids or purified from mammalian cells. The invention provides isolated IKK-β hybridization probes and primers capable of specifically hybridizing with the disclosed IKK-β gene, IKK-β-specific binding agents such as specific antibodies, and methods of making and using the subject compositions in diagnosis (e.g. genetic hybridization screens for IKK-β transcripts), therapy (e.g. IKK-β kinase inhibitors to inhibit TNF signal transduction) and in the biopharmaceutical industry (e.g. as immunogens, reagents for isolating other transcriptional regulators, reagents for screening chemical libraries for lead pharmacological agents, etc.).

DETAILED DESCRIPTION OF THE INVENTION

The nucleotide sequence of a natural cDNA encoding a human IKK-β polypeptide is shown as SEQ ID NO: 1, and the full conceptual translate is shown as SEQ ID NO: 2. The IKK-β polypeptides of the invention include incomplete translates of SEQ ID NO: 1 and deletion mutants of SEQ ID NO: 2, which translates and deletion mutants have IKK-β-specific amino acid sequence and binding specificity or function.

The IKK-β polypeptide domains of the invention have amino acid sequences distinguishable from IKK-α, generally at least 11, preferably at least 21, more preferably at least 31 consecutive residues of SEQ ID NO: 2 and provide IKK-β domain specific activity or function, such as IKK-β-specific kinase or kinase inhibitory activity, NIK-binding or binding inhibitory activity, IκB-binding or binding inhibitory activity, NFκB activating or inhibitory activity or antibody binding. Preferred domains phosphorylate at least one and preferably both the serine 32 and 36 of IκB (Verma, I. M., et al. (1995)).

IKK-β-specific activity or function may be determined by convenient in vitro, cell-based, or in vivo assays: e.g. in vitro binding assays, cell culture assays, in animals (e.g. gene therapy, transgenics, etc.), etc. Binding assays encompass any assay where the molecular interaction of an IKK-β polypeptide with a binding target is evaluated. The binding target may be a natural intracellular binding target such as an IKK-β substrate, a IKK-β regulating protein or other regulator that directly modulates IKK-β activity or its localization; or non-natural binding target such a specific immune protein such as an antibody, or an IKK-β specific agent such as those identified in screening assays such as described below. IKK-β-binding specificity may assayed by kinase activity or binding equilibrium constants (usually at least about 10⁷ M⁻¹, preferably at least about 10⁸ M⁻¹, more preferably at least about 10⁹ M⁻¹), by the ability of the subject polypeptide to function as negative mutants in IKK-β-expressing cells, to elicit IKK-β specific antibody in a heterologous host (e.g a rodent or rabbit), etc. In any event, the IKK-β binding specificity of the subject IKK-β polypeptides necessarily distinguishes IKK-α (SEQ ID NO: 4).

The claimed IKK-β polypeptides are isolated or pure: an "isolated" polypeptide is unaccompanied by at least some of the material with which it is associated in its natural state, preferably constituting at least about 0.5%, and more preferably at least about 5% by weight of the total polypeptide in a given sample and a pure polypeptide constitutes at least about 90%, and preferably at least about 99% by weight of the total polypeptide in a given sample. The IKK-β polypeptides and polypeptide domains may be synthesized, produced by recombinant technology, or purified from mammalian, preferably human cells. A wide variety of molecular and biochemical methods are available for biochemical synthesis, molecular expression and purification of the subject compositions, see e.g. Molecular Cloning, A Laboratory Manual (Sambrook, et al. Cold Spring Harbor Laboratory), Current Protocols in Molecular Biology (Eds. Ausubel, et al., Greene Publ. Assoc., Wiley-Interscience, NY) or that are otherwise known in the art.

The invention provides binding agents specific to the subject kinase proteins including substrates, agonists, antagonists, natural intracellular binding targets, etc., methods of identifying and making such agents, and their use in diagnosis, therapy and pharmaceutical development. For example, specific binding agents are useful in a variety of diagnostic and therapeutic applications, especially where disease or disease prognosis is associated with improper utilization of a pathway involving the subject proteins, e.g. NF-κB activation. Novel IKK-β-specific binding agents include IKK-β-specific receptors, such as somatically recombined polypeptide receptors like specific antibodies or T-cell antigen receptors (see, e.g Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory) and other natural intracellular binding agents identified with assays such as one-, two- and three-hybrid screens, non-natural intracellular binding agents identified in screens of chemical libraries such as described below, etc. Agents of particular interest modulate IKK-β function, e.g. IKK-β-dependent transcriptional activation. For example, a wide variety of inhibitors of IKK-β IκB kinase activity may be used to regulate signal transduction involving IκB. Exemplary IKK-β IκB kinase inhibitors include known classes of serine/threonine kinase (e.g. PKC) inhibitors such as competitive inhibitors of ATP and substrate binding, antibiotics, IKK-β-derived peptide inhibitors, etc., see Tables 1 and 2. IKK-β specificity and activity are readily quantified in high throughput kinase assays using panels of protein kinases (see cited references and Examples).

Preferred inhibitors include natural compounds such as staurosporine (Omura S, et al. J Antibiot (Tokyo) 1995 July;48(7):535-48), produced by a marine organism, and synthetic compounds such as PD 153035, which also potently inhibits the EGF receptor protein kinase (Fry D W et al. Science 1994 Aug. 19;265(5175):1093-5). Members of the tyrphostin family of synthetic protein kinase inhibitors are also useful; these include compounds which are pure ATP competitors, compounds which are pure substrate competitors, and compounds which are mixed competitors: compete with both ATP and substrate (Levitzki A and Gazit A, Science 1995 Mar 24;267(5205):1782-8). Additional IKK-β inhibitors include peptide-based substrate competitors endogenously made by the mammalian cell, e.g. PKI (protein kinase inhibitor, Seasholtz A F et al., Proc Natl Acad Sci USA 1995 Feb. 28;92(5):1734-8), or proteins inhibiting cdc kinases (Correa-Bordes J and Nurse P, Cell 1995 Dec. 15;83(6):1001-9). Additional small peptide based substrate competitive kinase inhibitors and allosteric inhibitors (inhibitory mechanisms independent of ATP or substrate competition) are readily generated by established methods (Hvalby O, et al. Proc Natl Acad Sci USA 1994 May 24;91(11):4761-5; Barja P, et al., Cell Immunol 1994 January;153(1):28-38; Villar-Palasi C, Biochim Biophys Acta 1994 Dec. 30;1224(3):384-8; Liu W Z, et al., Biochemistry 1994 Aug. 23;33(33):10120-6).

                  TABLE I                                                          ______________________________________                                         Selected Small Molecule IKK-β Kinase Inhibitors                           Inhibitors  Citations                                                          ______________________________________                                         HA-100.sup.1                                                                                1.   Hagiwara, M,. et al. Mol. Pharmacol.                                           32:7 (1987)                                                  Chelerythrine.sup.2                                                                         2.   Herbert, J. M., et al. Biochem Biophys                                         Res Com 172:993 (1990)                                       Staurosporine.sup.3,4,5                                                                     3.   Schachtele, C., et al. Biochem Biophys                                         Res Com 151:542 (1988)                                       Calphostin C.sup.6,7,8,9                                                                    4.   Tamaoki, T., et al. Biochem Biophys Res                                        Com 135:397 (1986)                                           K-252b.sup.10                                                                               5.   Tischler, A. S., et al. J. Neurochemistry                                      55:1159 (1990)                                               PKC 19-36.sup.11                                                                            6.   Bruns, R. F., et al. Biochem Biophys Res                                       Com 176:288 (1991)                                           Iso-H7.sup.12                                                                               7.   Kobayashi, E., et al. Biochem Biophys Res                                      Com 159:548 (1989)                                           PKC 19-31    8.   Tamaoki, T., et al Adv 2nd Mass Phospho-                                       protein Res 24:497 (1990)                                    H-7.sup.13,3,14                                                                             9.   Tamaoki, T., et al. Biotechnology 8:732                                        (1990)                                                       H-89.sup.15 10.   Yasuzawa, T. J. Antibiotics 39:1972                                            (1986)                                                       KT5720.sup.16                                                                              11.   House, C., et al. Science 238:1726                                             (1987)                                                       cAMP-depPKinhib.sup.17                                                                     12.   Quick, J., et al. Biochem. Biophys. Res.                                       Com. 167:657 (1992)                                          A-3.sup.18  13.   Bouli, N. M. and Davis, M. Brain Res.                                          525:198 (1990)                                               HA1004.sup.19,20                                                                           14.   Takahashi, I., et al. J. Pharmacol. Exp. Ther.                                 255:1218 (1990)                                              K-252a.sup.16,5                                                                            15.   Chijiwa, T., et al. J. Biol. Chem. 265:5267                                    (1990)                                                       KT5823.sup.16                                                                              16.   Kase, H., et al. Biochem. Biophys. Res.                                        Com. 142:436 (1987)                                          ML-9.sup.21 17.   Cheng, H. C., et al. J. Biol. Chem. 261:989                                    (1986)                                                       KT5926.sup.22                                                                              18.   Inagaki, M., et al. Mol. Pharmacol. 29:577                                     (1986)                                                                   19.   Asano, T. and Hidaka, H. J Pharmaco. Exp                                       Ther 231:141 (1984)                                                      20.   Hidaka, H., et al. Biochemistry 23:5036                                        (1984)                                                                   21.   Nagatsu, T., et al. Biochem Biophys Res                                        Com 143:1045 (1987)                                                      22.   Nakanishi, S., et al. Mol. Pharmacol.                                          37:482 (1990)                                                ______________________________________                                    

                  TABLE II                                                         ______________________________________                                         Selected Peptidyl IKK-β Kinase Inhibitors                                 ______________________________________                                         hIκBα, residues 24-39, 32Ala                                                            hIKK-β, Δ5-203                                 hIκBα, residues 29-47, 36Ala                                                            hIKK-β, Δ1-178                                 hIκBα, residues 26-46, 32/36Ala                                                         hIKK-β, Δ368-756                               hIκBβ, residues 25-38, 32Ala                                                             hIKK-β, Δ460-748                               hIκBβ, residues 30-41, 36Ala                                                             hIKK-α, Δ1-289                                hIκBβ, residues 26-46, 32/36Ala                                                          hIKK-α, Δ12-219                               hIκBε, residues 24-40, 32Ala                                                          hIKK-α, Δ307-745                              hIκBε, residues 31-50, 36Ala                                                          hIKK-α, Δ319-644                              hIκBε, residues 27-44, 32/36Ala                                  ______________________________________                                    

Accordingly, the invention provides methods for modulating signal transduction involving IκB in a cell comprising the step of modulating IKK-β kinase activity, e.g. by contacting the cell with a serine/threonine kinase inhibitor. The cell may reside in culture or in situ, i.e. within the natural host. Preferred inhibitors are orally active in mammalian hosts. For diagnostic uses, the inhibitors or other IKK-β binding agents are frequently labeled, such as with fluorescent, radioactive, chemiluminescent, or other easily detectable molecules, either conjugated directly to the binding agent or conjugated to a probe specific for the binding agent.

The amino acid sequences of the disclosed IKK-β polypeptides are used to back-translate IKK-β polypeptide-encoding nucleic acids optimized for selected expression systems (Holler et al. (1993) Gene 136, 323-328; Martin et al. (1995) Gene 154, 150-166) or used to generate degenerate oligonucleotide primers and probes for use in the isolation of natural IKK-β-encoding nucleic acid sequences ("GCG" software, Genetics Computer Group, Inc, Madison Wis.). IKK-β-encoding nucleic acids used in IKK-β-expression vectors and incorporated into recombinant host cells, e.g. for expression and screening, transgenic animals, e.g. for functional studies such as the efficacy of candidate drugs for disease associated with IKK-β-modulated cell function, etc.

The invention also provides nucleic acid hybridization probes and replication/amplification primers having a IKK-β cDNA specific sequence contained in SEQ ID NO: 1 and sufficient to effect specific hybridization thereto (i.e. specifically hybridize with SEQ ID NO: 1 in the presence of IKK-α cDNA, SEQ ID NO: 3). Such primers or probes are at least 12, preferably at least 24, more preferably at least 36 and most preferably at least 96 bases in length. Demonstrating specific hybridization generally requires stringent conditions, for example, hybridizing in a buffer comprising 30% formamide in 5×SSPE (0.18M NaCl, 0.01M NaPO₄, pH7.7, 0.001M EDTA) buffer at a temperature of 42° C. and remaining bound when subject to washing at 42° C. with 0.2×SSPE; preferably hybridizing in a buffer comprising 50% formamide in 5×SSPE buffer at a temperature of 42° C. and remaining bound when subject to washing at 42° C. with 0.2×SSPE buffer at 42° C. IKK-β nucleic acids can also be distinguished using alignment algorithms, such as BLASTX (Altschul et al. (1990) Basic Local Alignment Search Tool, J Mol Biol 215, 403-410).

The subject nucleic acids are of synthetic/non-natural sequences and/or are isolated, i.e. unaccompanied by at least some of the material with which it is associated in its natural state, preferably constituting at least about 0.5%, preferably at least about 5% by weight of total nucleic acid present in a given fraction, and usually recombinant, meaning they comprise a non-natural sequence or a natural sequence joined to nucleotide(s) other than that which it is joined to on a natural chromosome. Recombinant nucleic acids comprising the nucleotide sequence of SEQ ID NO: 1 or fragments thereof, contain such sequence or fragment at a terminus, immediately flanked by a sequence other than that which it is joined to on a natural chromosome, or flanked by a native flanking region fewer than 10 kb, preferably fewer than 2 kb, which is at a terminus or is immediately flanked by a sequence other than that which it is joined to on a natural chromosome. While the nucleic acids are usually RNA or DNA, it is often advantageous to use nucleic acids comprising other bases or nucleotide analogs to provide modified stability, etc.

The subject nucleic acids find a wide variety of applications including use as translatable transcripts, hybridization probes, PCR primers, diagnostic nucleic acids, etc.; use in detecting the presence of IKK-β genes and gene transcripts and in detecting or amplifying nucleic acids encoding additional IKK-β homologs and structural analogs. In diagnosis, IKK-β hybridization probes find use in identifying wild-type and mutant IKK-β alleles in clinical and laboratory samples. Mutant alleles are used to generate allele-specific oligonucleotide (ASO) probes for high-throughput clinical diagnoses. In therapy, therapeutic IKK-β nucleic acids are used to modulate cellular expression or intracellular concentration or availability of active IKK-β.

The invention provides efficient methods of identifying agents, compounds or lead compounds for agents active at the level of a IKK-β modulatable cellular function. Generally, these screening methods involve assaying for compounds which modulate IKK-β interaction with a natural IKK-β binding target, in particular, IKK-β phosphorylation of IκB-derived substrates, particularly IκB and NIK substrates. A wide variety of assays for binding agents are provided including labeled in vitro protein-protein binding assays, immunoassays, cell based assays, etc. The methods are amenable to automated, cost-effective high throughput screening of chemical libraries for lead compounds. Identified reagents find use in the pharmaceutical industries for animal and human trials; for example, the reagents may be derivatized and rescreened in in vitro and in vivo assays to optimize activity and minimize toxicity for pharmaceutical development.

In vitro binding assays employ a mixture of components including an IKK-β polypeptide, which may be part of a fusion product with another peptide or polypeptide, e.g. a tag for detection or anchoring, etc. The assay mixtures comprise a natural intracellular IKK-β binding target. In a particular embodiment, the binding target is a substrate comprising IκB serines 32 and/or 36. Such substrates comprise a IκBα, β or ε peptide including the serine 32 and/or 36 residue and at least 5, preferably at least 10, and more preferably at least 20 naturally occurring immediately flanking residues on each side (e.g. for serine 36 peptides, residues 26-46, 22-42, or 12-32 or 151-171 for IκBα, β or ε -derived substrates, respectively). While native full-length binding targets may be used, it is frequently preferred to use portions (e.g. peptides) thereof so long as the portion provides binding affinity and avidity to the subject IKK-β polypeptide conveniently measurable in the assay. The assay mixture also comprises a candidate pharmacological agent. Candidate agents encompass numerous chemical classes, though typically they are organic compounds; preferably small organic compounds and are obtained from a wide variety of sources including libraries of synthetic or natural compounds. A variety of other reagents may also be included in the mixture. These include reagents like ATP or ATP analogs (for kinase assays), salts, buffers, neutral proteins, e.g. albumin, detergents, protease inhibitors, nuclease inhibitors, antimicrobial agents, etc. may be used.

The resultant mixture is incubated under conditions whereby, but for the presence of the candidate pharmacological agent, the IKK-β polypeptide specifically binds the cellular binding target, portion or analog with a reference binding affinity. The mixture components can be added in any order that provides for the requisite bindings and incubations may be performed at any temperature which facilitates optimal binding. Incubation periods are likewise selected for optimal binding but also minimized to facilitate rapid, high-throughput screening.

After incubation, the agent-biased binding between the IKK-β polypeptide and one or more binding targets is detected by any convenient way. For IKK-β kinase assays, `binding` is generally detected by a change in the phosphorylation of a IKK-β substrate. In this embodiment, kinase activity may quantified by the transfer to the substrate of a labeled phosphate, where the label may provide for direct detection as radioactivity, luminescence, optical or electron density, etc. or indirect detection such as an epitope tag, etc. A variety of methods may be used to detect the label depending on the nature of the label and other assay components, e.g. through optical or electron density, radiative emissions, nonradiative energy transfers, etc. or indirectly detected with antibody conjugates, etc.

A difference in the binding affinity of the IKK-β polypeptide to the target in the absence of the agent as compared with the binding affinity in the presence of the agent indicates that the agent modulates the binding of the IKK-β polypeptide to the IKK-β binding target. Analogously, in the cell-based assay also described below, a difference in IKK-β-dependent transcriptional activation in the presence and absence of an agent indicates the agent modulates IKK-β function. A difference, as used herein, is statistically significant and preferably represents at least a 50%, more preferably at least a 90% difference.

The following experimental section and examples are offered by way of illustration and not by way of limitation.

EXPERIMENTAL

Identification of IKK-β

To investigate the mechanism of NIK-mediated NF-κB activation, we initially identified proteins that associate directly with NIK by yeast two-hybrid protein interaction cloning (Fields and Song, 1989). An expression vector was generated that encodes NIK fused to the DNA-binding domain of the yeast transcription factor GAL4. This vector was used as bait in a two-hybrid screen of a human B cell cDNA library. From approximately six million transformants, eight positive clones were obtained, as determined by activation of his and lacZ reporter genes. Of these clones, three encoded a member of the TRAF family, TRAF3 (Hu et al., 1994; Cheng et al., 1995; Mosialos et al., 1995; Sato et al., 1995) and one encoded a novel protein we call IKK-α. Retransformation into yeast cells verified the interaction between NIK and IKK-α. A full-length human IKK-α clone was isolated by screening a Jurkat cDNA library with a probe generated from the 5'-end of the IKK-α two-hybrid clone. IKK-α comprises an N-terminal serine-threonine kinase catalytic domain, a C-terminal helix-loop-helix domain and a leucine zipper-like amphipathic α-helix juxtaposed in between the helix-loop-helix and kinase domain.

To identify potential IKK-α-related genes, we searched a public database of human expressed sequence tags (ESTs). We identified two ESTs (W68756 and AA128064), which we determined were capable of encoding distinct peptides with sequence similarity with IKK-α. IKK-α-related cDNA was cloned by probing a Jurkat cDNA library (human T cell) with an oligonucleotide probe corresponding to sequence from one of the ESTs. Sequence analysis demonstrated that the two ESTs included different fragments of the same gene. The longest cDNA clones obtained had a ˜3.2 kb insert (SEQ ID NO: 1) and an open reading frame of 756 amino acids (SEQ ID NO: 2). We have designed the protein encoded by this cDNA as IKK-β.

Interaction of IKK-β and NIK in Human Cells

The interaction of IKK-β with NIK was confirmed in mammalian cell coimmunoprecipitation assays. Human IKK-β containing an C-terminal Flag epitope tag was transiently coexpressed in 293 human embryonic kidney cells with Myc epitope-tagged NIK. Cell lysates were immunoprecipitated using a monoclonal antibody against the Flag epitope, and coprecipitating NIK was detected by immunoblot analysis with an anti-Myc monoclonal antibody. In this assay, IKK-β was able to coprecipitate NIK confirming the interaction between both proteins as detected for IKK-α by yeast two-hybrid analysis. Also, a deletion mutant IKK-β protein lacking most of the N-terminal kinase domain (IKK-β.sub.(Δ5-203, i.e. 1-4 & 204-756)) was able to associate with NIK, indicating that the α-helical C-terminal half of IKK-β mediates the interaction with NIK.

Effect of IKK-β and IKK-β Mutants on NF-κB Activation

To investigate a possible role for IKK-β in NF-κB activation, we examined if transient overexpression of IKK-β might activate an NF-κB-dependent reporter gene. An E-selectin-luciferase reporter construct (Schindler and Baichwal, 1994) and a IKK-β expression vector were cotransfected into HeLa cells. IKK-β expression activated the reporter gene in a dose-dependent manner, with a maximal induction of luciferase activity of about 20-fold compared to vector control. Similar results were obtained in 293 cells, where IKK-β overexpression induced reporter gene activity approximately 20-fold. Thus, IKK-β induces NF-κB activation when overexpressed.

We next determined the effect of overexpression of kinase-inactive IKK-β.sub.(Δ5-203) that still associates with NIK on signal-induced NF-κB activation in reporter gene assays in 293 cells. Overexpression of IKK-β.sub.(Δ5-203) blocked TNF- and IL-1-induced reporter gene activation similar to overexpression of NIK.sub.(624-947). IKK-β.sub.(Δ5-203) was also found to inhibited NF-κB-dependent reporter gene activity elicited by overexpression of TRAF2, TRAF6 and NIK. Taken together these results demonstrate that a catalytically inactive IKK-β mutant is a dominant-negative inhibitor of TNF-, IL-1, TRAF- and NIK-induced NF-κB activation. This indicates that IKK-β functions as a common mediator of NF-κB activation by TNF and IL-1 downstream of NIK.

Parenthetical References

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EXAMPLES

1. Protocol for at IKK-β-IκBα phosphorylation assay.

A. Reagents:

Neutralite Avidin: 20 μg/ml in PBS.

kinase: 10⁻⁸ -10⁻⁵ M IKK-β (SEQ ID NO: 2) at 20 μg/ml in PBS.

substrate: 10⁻⁷ -10⁻⁴ M biotinylated substrate (21 residue peptide consisting of residues 26-46 of human IκBα) at 40 μg/ml in PBS.

Blocking buffer: 5% BSA, 0.5% Tween 20 in PBS; 1 hour at room temperature.

Assay Buffer: 100 mM KCl, 10 mM MgCl₂, 1 mM MnCl₂, 20 mM HEPES pH 7.4, 0.25 mM EDTA, 1% glycerol, 0.5% NP-40, 50 mM BME, 1 mg/ml BSA, cocktail of protease inhibitors.

³² P!γ-ATP 10x stock: 2×10⁻⁵ M cold ATP with 100 μCi ³² P!γ-ATP. Place in the 4° C. microfridge during screening.

Protease inhibitor cocktail (1000×): 10 mg Trypsin Inhibitor (BMB # 109894), 10 mg Aprotinin (BMB # 236624), 25 mg Benzamidine (Sigma # B-6506), 25 mg Leupeptin (BMB # 1017128), 10 mg APMSF (BMB # 917575), and 2 mM NaVo₃ (Sigma # S-6508) in 10 ml of PBS.

B. Preparation of assay plates:

Coat with 120 μl of stock N Avidin per well overnight at 4° C.

Wash 2 times with 200 μl PBS.

Block with 150 μl of blocking buffer.

Wash 2 times with 200 μl PBS.

C. Assay:

Add 40 μl assay buffer/well.

Add 40 μl biotinylated substrate (2-200 pmoles/40 ul in assay buffer)

Add 40 μl kinase (0.1-10 pmoles/40 ul in assay buffer)

Add 10 μl compound or extract.

Add 10 μl ³² P!γ-ATP 10x stock.

Shake at 25° C. for 15 minutes.

Incubate additional 45 minutes at 25° C.

Stop the reaction by washing 4 times with 200 μl PBS.

Add 150 μl scintillation cocktail.

Count in Topcount.

D. Controls for all assays (located on each plate):

a. Non-specific binding

b. cold ATP at 80% inhibition.

2. Protocol for high throughput IKK-β-NIK binding assay.

A. Reagents:

Neutralite Avidin: 20 μg/ml in PBS.

Blocking buffer: 5% BSA, 0.5% Tween 20 in PBS; 1 hour at room temperature.

Assay Buffer: 100 mM KCl, 20 mM HEPES pH 7.6, 1 mM MgCl₂, 1% glycerol, 0.5% NP-40, 50 mM β-mercaptoethanol, 1 mg/ml BSA, cocktail of protease inhibitors.

³³ P IKK-β polypeptide 10x stock: 10⁻⁸ -10⁻⁶ M "cold" IKK-β supplemented with 200,000-250,000 cpm of labeled IKK-β (Beckman counter). Place in the 4° C. microfridge during screening.

Protease inhibitor cocktail (1000X): 10 mg Trypsin Inhibitor (BMB # 109894), 10 mg Aprotinin (BMB # 236624), 25 mg Benzamidine (Sigma # B-6506), 25 mg Leupeptin (BMB # 1017128), 10 mg APMSF (BMB # 917575), and 2 mM NaVO₃ (Sigma # S-6508) in 10 ml of PBS.

NIK: 10⁻⁷ -10⁻⁵ M biotinylated NIK in PBS.

B. Preparation of assay plates:

Coat with 120 μl of stock N-Avidin per well overnight at 4° C.

Wash 2 times with 200 μl PBS.

Block with 150 μl of blocking buffer.

Wash 2 times with 200 μl PBS.

C. Assay:

Add 40 μl assay buffer/well.

Add 10 μl compound or extract.

Add 10 μl ³³ P-IKK-β (20-25,000 cpm/0.1-10 pmoles/well=10⁻⁹ -10⁻⁷ M final conc).

Shake at 25° C. for 15 minutes.

Incubate additional 45 minutes at 25° C.

Add 40 μM biotinylated NIK (0.1-10 pmoles/40 ul in assay buffer)

Incubate 1 hour at room temperature.

Stop the reaction by washing 4 times with 200 μM PBS.

Add 150 μM scintillation cocktail.

Count in Topcount.

D. Controls for all assays (located on each plate):

a. Non-specific binding

b. Soluble (non-biotinylated NIK) at 80% inhibition.

All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

    __________________________________________________________________________     SEQUENCE LISTING                                                               (1) GENERAL INFORMATION:                                                       (iii) NUMBER OF SEQUENCES: 4                                                   (2) INFORMATION FOR SEQ ID NO:1:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 2268 base pairs                                                    (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                        ATGAGCTGGTCACCTTCCCTGACAACGCAGACATGTGGGGCCTGGGAAATGAAAGAGCGC60                 CTTGGGACAGGGGGATTTGGAAATGTCATCCGATGGCACAATCAGGAAACAGGTGAGCAG120                ATTGCCATCAAGCAGTGCCGGCAGGAGCTCAGCCCCCGGAACCGAGAGCGGTGGTGCCTG180                GAGATCCAGATCATGAGAAGGCTGACCCACCCCAATGTGGTGGCTGCCCGAGATGTCCCT240                GAGGGGATGCAGAACTTGGCGCCCAATGACCTGCCCCTGCTGGCCATGGAGTACTGCCAA300                GGAGGAGATCTCCGGAAGTACCTGAACCAGTTTGAGAACTGCTGTGGTCTGCGGGAAGGT360                GCCATCCTCACCTTGCTGAGTGACATTGCCTCTGCGCTTAGATACCTTCATGAAAACAGA420                ATCATCCATCGGGATCTAAAGCCAGAAAACATCGTCCTGCAGCAAGGAGAACAGAGGTTA480                ATACACAAAATTATTGACCTAGGATATGCCAAGGAGCTGGATCAGGGCAGTCTTTGCACA540                TCATTCGTGGGGACCCTGCAGTACCTGGCCCCAGAGCTACTGGAGCAGCAGAAGTACACA600                GTGACCGTCGACTACTGGAGCTTCGGCACCCTGGCCTTTGAGTGCATCACGGGCTTCCGG660                CCCTTCCTCCCCAACTGGCAGCCCGTGCAGTGGCATTCAAAAGTGCGGCAGAAGAGTGAG720                GTGGACATTGTTGTTAGCGAAGACTTGAATGGAACGGTGAAGTTTTCAAGCTCTTTACCC780                TACCCCAATAATCTTAACAGTGTCCTGGCTGAGCGACTGGAGAAGTGGCTGCAACTGATG840                CTGATGTGGCACCCCCGACAGAGGGGCACGGATCCCACGTATGGGCCCAATGGCTGCTTC900                AAGGCCCTGGATGACATCTTAAACTTAAAGCTGGTTCATATCTTGAACATGGTCACGGGC960                ACCATCCACACCTACCCTGTGACAGAGGATGAGAGTCTGCAGAGCTTGAAGGCCAGAATC1020               CAACAGGACACGGGCATCCCAGAGGAGGACCAGGAGCTGCTGCAGGAAGCGGGCCTGGCG1080               TTGATCCCCGATAAGCCTGCCACTCAGTGTATTTCAGACGGCAAGTTAAATGAGGGCCAC1140               ACATTGGACATGGATCTTGTTTTTCTCTTTGACAACAGTAAAATCACCTATGAGACTCAG1200               ATCTCCCCACGGCCCCAACCTGAAAGTGTCAGCTGTATCCTTCAAGAGCCCAAGAGGAAT1260               CTCGCCTTCTTCCAGCTGAGGAAGGTGTGGGGCCAGGTCTGGCACAGCATCCAGACCCTG1320               AAGGAAGATTGCAACCGGCTGCAGCAGGGACAGCGAGCCGCCATGATGAATCTCCTCCGA1380               AACAACAGCTGCCTCTCCAAAATGAAGAATTCCATGGCTTCCATGTCTCAGCAGCTCAAG1440               GCCAAGTTGGATTTCTTCAAAACCAGCATCCAGATTGACCTGGAGAAGTACAGCGAGCAA1500               ACCGAGTTTGGGATCACATCAGATAAACTGCTGCTGGCCTGGAGGGAAATGGAGCAGGCT1560               GTGGAGCTCTGTGGGCGGGAGAACGAAGTGAAACTCCTGGTAGAACGGATGATGGCTCTG1620               CAGACCGACATTGTGGACTTACAGAGGAGCCCCATGGGCCGGAAGCAGGGGGGAACGCTG1680               GACGACCTAGAGGAGCAAGCAAGGGAGCTGTACAGGAGACTAAGGGAAAAACCTCGAGAC1740               CAGCGAACTGAGGGTGACAGTCAGGAAATGGTACGGCTGCTGCTTCAGGCAATTCAGAGC1800               TTCGAGAAGAAAGTGCGAGTGATCTATACGCAGCTCAGTAAAACTGTGGTTTGCAAGCAG1860               AAGGCGCTGGAACTGTTGCCCAAGGTGGAAGAGGTGGTGAGCTTAATGAATGAGGATGAG1920               AAGACTGTTGTCCGGCTGCAGGAGAAGCGGCAGAAGGAGCTCTGGAATCTCCTGAAGATT1980               GCTTGTAGCAAGGTCCGTGGTCCTGTCAGTGGAAGCCCGGATAGCATGAATGCCTCTCGA2040               CTTAGCCAGCCTGGGCAGCTGATGTCTCAGCCCTCCACGGCCTCCAACAGCTTACCTGAG2100               CCAGCCAAGAAGAGTGAAGAACTGGTGGCTGAAGCACATAACCTCTGCACCCTGCTAGAA2160               AATGCCATACAGGACACTGTGAGGGAACAAGACCAGAGTTTCACGGCCCTAGACTGGAGC2220               TGGTTACAGACGGAAGAAGAAGAGCACAGCTGCCTGGAGCAGGCCTCA2268                           (2) INFORMATION FOR SEQ ID NO:2:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 756 amino acids                                                    (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                        MetSerTrpSerProSerLeuThrThrGlnThrCysGlyAlaTrpGlu                               151015                                                                         MetLysGluArgLeuGlyThrGlyGlyPheGlyAsnValIleArgTrp                               202530                                                                         HisAsnGlnGluThrGlyGluGlnIleAlaIleLysGlnCysArgGln                               354045                                                                         GluLeuSerProArgAsnArgGluArgTrpCysLeuGluIleGlnIle                               505560                                                                         MetArgArgLeuThrHisProAsnValValAlaAlaArgAspValPro                               65707580                                                                       GluGlyMetGlnAsnLeuAlaProAsnAspLeuProLeuLeuAlaMet                               859095                                                                         GluTyrCysGlnGlyGlyAspLeuArgLysTyrLeuAsnGlnPheGlu                               100105110                                                                      AsnCysCysGlyLeuArgGluGlyAlaIleLeuThrLeuLeuSerAsp                               115120125                                                                      IleAlaSerAlaLeuArgTyrLeuHisGluAsnArgIleIleHisArg                               130135140                                                                      AspLeuLysProGluAsnIleValLeuGlnGlnGlyGluGlnArgLeu                               145150155160                                                                   IleHisLysIleIleAspLeuGlyTyrAlaLysGluLeuAspGlnGly                               165170175                                                                      SerLeuCysThrSerPheValGlyThrLeuGlnTyrLeuAlaProGlu                               180185190                                                                      LeuLeuGluGlnGlnLysTyrThrValThrValAspTyrTrpSerPhe                               195200205                                                                      GlyThrLeuAlaPheGluCysIleThrGlyPheArgProPheLeuPro                               210215220                                                                      AsnTrpGlnProValGlnTrpHisSerLysValArgGlnLysSerGlu                               225230235240                                                                   ValAspIleValValSerGluAspLeuAsnGlyThrValLysPheSer                               245250255                                                                      SerSerLeuProTyrProAsnAsnLeuAsnSerValLeuAlaGluArg                               260265270                                                                      LeuGluLysTrpLeuGlnLeuMetLeuMetTrpHisProArgGlnArg                               275280285                                                                      GlyThrAspProThrTyrGlyProAsnGlyCysPheLysAlaLeuAsp                               290295300                                                                      AspIleLeuAsnLeuLysLeuValHisIleLeuAsnMetValThrGly                               305310315320                                                                   ThrIleHisThrTyrProValThrGluAspGluSerLeuGlnSerLeu                               325330335                                                                      LysAlaArgIleGlnGlnAspThrGlyIleProGluGluAspGlnGlu                               340345350                                                                      LeuLeuGlnGluAlaGlyLeuAlaLeuIleProAspLysProAlaThr                               355360365                                                                      GlnCysIleSerAspGlyLysLeuAsnGluGlyHisThrLeuAspMet                               370375380                                                                      AspLeuValPheLeuPheAspAsnSerLysIleThrTyrGluThrGln                               385390395400                                                                   IleSerProArgProGlnProGluSerValSerCysIleLeuGlnGlu                               405410415                                                                      ProLysArgAsnLeuAlaPhePheGlnLeuArgLysValTrpGlyGln                               420425430                                                                      ValTrpHisSerIleGlnThrLeuLysGluAspCysAsnArgLeuGln                               435440445                                                                      GlnGlyGlnArgAlaAlaMetMetAsnLeuLeuArgAsnAsnSerCys                               450455460                                                                      LeuSerLysMetLysAsnSerMetAlaSerMetSerGlnGlnLeuLys                               465470475480                                                                   AlaLysLeuAspPhePheLysThrSerIleGlnIleAspLeuGluLys                               485490495                                                                      TyrSerGluGlnThrGluPheGlyIleThrSerAspLysLeuLeuLeu                               500505510                                                                      AlaTrpArgGluMetGluGlnAlaValGluLeuCysGlyArgGluAsn                               515520525                                                                      GluValLysLeuLeuValGluArgMetMetAlaLeuGlnThrAspIle                               530535540                                                                      ValAspLeuGlnArgSerProMetGlyArgLysGlnGlyGlyThrLeu                               545550555560                                                                   AspAspLeuGluGluGlnAlaArgGluLeuTyrArgArgLeuArgGlu                               565570575                                                                      LysProArgAspGlnArgThrGluGlyAspSerGlnGluMetValArg                               580585590                                                                      LeuLeuLeuGlnAlaIleGlnSerPheGluLysLysValArgValIle                               595600605                                                                      TyrThrGlnLeuSerLysThrValValCysLysGlnLysAlaLeuGlu                               610615620                                                                      LeuLeuProLysValGluGluValValSerLeuMetAsnGluAspGlu                               625630635640                                                                   LysThrValValArgLeuGlnGluLysArgGlnLysGluLeuTrpAsn                               645650655                                                                      LeuLeuLysIleAlaCysSerLysValArgGlyProValSerGlySer                               660665670                                                                      ProAspSerMetAsnAlaSerArgLeuSerGlnProGlyGlnLeuMet                               675680685                                                                      SerGlnProSerThrAlaSerAsnSerLeuProGluProAlaLysLys                               690695700                                                                      SerGluGluLeuValAlaGluAlaHisAsnLeuCysThrLeuLeuGlu                               705710715720                                                                   AsnAlaIleGlnAspThrValArgGluGlnAspGlnSerPheThrAla                               725730735                                                                      LeuAspTrpSerTrpLeuGlnThrGluGluGluGluHisSerCysLeu                               740745750                                                                      GluGlnAlaSer                                                                   755                                                                            (2) INFORMATION FOR SEQ ID NO:3:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 2238 base pairs                                                    (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                        ATGGAGCGGCCCCCGGGGCTGCGGCCGGGCGCGGGCGGGCCCTGGGAGATGCGGGAGCGG60                 CTGGGCACCGGCGGCTTCGGGAACGTCTGTCTGTACCAGCATCGGGAACTTGATCTCAAA120                ATAGCAATTAAGTCTTGTCGCCTAGAGCTAAGTACCAAAAACAGAGAACGATGGTGCCAT180                GAAATCCAGATTATGAAGAAGTTGAACCATGCCAATGTTGTAAAGGCCTGTGATGTTCCT240                GAAGAATTGAATATTTTGATTCATGATGTGCCTCTTCTAGCAATGGAATACTGTTCTGGA300                GGAGATCTCCGAAAGCTGCTCAACAAACCAGAAAATTGTTGTGGACTTAAAGAAAGCCAG360                ATACTTTCTTTACTAAGTGATATAGGGTCTGGGATTCGATATTTGCATGAAAACAAAATT420                ATACATCGAGATCTAAAACCTGAAAACATAGTTCTTCAGGATGTTGGTGGAAAGATAATA480                CATAAAATAATTGATCTGGGATATGCCAAAGATGTTGATCAAGGAAGTCTGTGTACATCT540                TTTGTGGGAACACTGCAGTATCTGGCCCCAGAGCTCTTTGAGAATAAGCCTTACACAGCC600                ACTGTTGATTATTGGAGCTTTGGGACCATGGTATTTGAATGTATTGCTGGATATAGGCCT660                TTTTTGCATCATCTGCAGCCATTTACCTGGCATGAGAAGATTAAGAAGAAGGATCCAAAG720                TGTATATTTGCATGTGAAGAGATGTCAGGAGAAGTTCGGTTTAGTAGCCATTTACCTCAA780                CCAAATAGCCTTTGTAGTTTAATAGTAGAACCCATGGAAAACTGGCTACAGTTGATGTTG840                AATTGGGACCCTCAGCAGAGAGGAGGACCTGTTGACCTTACTTTGAAGCAGCCAAGATGT900                TTTGTATTAATGGATCACATTTTGAATTTGAAGATAGTACACATCCTAAATATGACTTCT960                GCAAAGATAATTTCTTTTCTGTTACCACCTGATGAAAGTCTTCATTCACTACAGTCTCGT1020               ATTGAGCGTGAAACTGGAATAAATACTGGTTCTCAAGAACTTCTTTCAGAGACAGGAATT1080               TCTCTGGATCCTCGGAAACCAGCCTCTCAATGTGTTCTAGATGGAGTTAGAGGCTGTGAT1140               AGCTATATGGTTTATTTGTTTGATAAAAGTAAAACTGTATATGAAGGGCCATTTGCTTCC1200               AGAAGTTTATCTGATTGTGTAAATTATATTGTACAGGACAGCAAAATACAGCTTCCAATT1260               ATACAGCTGCGTAAAGTGTGGGCTGAAGCAGTGCACTATGTGTCTGGACTAAAAGAAGAC1320               TATAGCAGGCTCTTTCAGGGACAAAGGGCAGCAATGTTAAGTCTTCTTAGATATAATGCT1380               AACTTAACAAAAATGAAGAACACTTTGATCTCAGCATCACAACAACTGAAAGCTAAATTG1440               GAGTTTTTTCACAAAAGCATTCAGCTTGACTTGGAGAGATACAGCGAGCAGATGACGTAT1500               GGGATATCTTCAGAAAAAATGCTAAAAGCATGGAAAGAAATGGAAGAAAAGGCCATCCAC1560               TATGCTGAGGTTGGTGTCATTGGATACCTGGAGGATCAGATTATGTCTTTGCATGCTGAA1620               ATCATGGAGCTACAGAAGAGCCCCTATGGAAGACGTCAGGGAGACTTGATGGAATCTCTG1680               GAACAGCGTGCCATTGATCTATATAAGCAGTTAAAACACAGACCTTCAGATCACTCCTAC1740               AGTGACAGCACAGAGATGGTGAAAATCATTGTGCACACTGTGCAGAGTCAGGACCGTGTG1800               CTCAAGGAGCTGTTTGGTCATTTGAGCAAGTTGTTGGGCTGTAAGCAGAAGATTATTGAT1860               CTACTCCCTAAGGTGGAAGTGGCCCTCAGTAATATCAAAGAAGCTGACAATACTGTCATG1920               TTCATGCAGGGAAAAAGGCAGAAAGAAATATGGCATCTCCTTAAAATTGCCTGTACACAG1980               AGTTCTGCCCGGTCCCTTGTAGGATCCAGTCTAGAAGGTGCAGTAACCCCTCAGACATCA2040               GCATGGCTGCCCCCGACTTCAGCAGAACATGATCATTCTCTGTCATGTGTGGTAACTCCT2100               CAAGATGGGGAGACTTCAGCACAAATGATAGAAGAAAATTTGAACTGCCTTGGCCATTTA2160               AGCACTATTATTCATGAGGCAAATGAGGAACAGGGCAATAGTATGATGAATCTTGATTGG2220               AGTTGGTTAACAGAATGA2238                                                         (2) INFORMATION FOR SEQ ID NO:4:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 745 amino acids                                                    (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                        MetGluArgProProGlyLeuArgProGlyAlaGlyGlyProTrpGlu                               151015                                                                         MetArgGluArgLeuGlyThrGlyGlyPheGlyAsnValCysLeuTyr                               202530                                                                         GlnHisArgGluLeuAspLeuLysIleAlaIleLysSerCysArgLeu                               354045                                                                         GluLeuSerThrLysAsnArgGluArgTrpCysHisGluIleGlnIle                               505560                                                                         MetLysLysLeuAsnHisAlaAsnValValLysAlaCysAspValPro                               65707580                                                                       GluGluLeuAsnIleLeuIleHisAspValProLeuLeuAlaMetGlu                               859095                                                                         TyrCysSerGlyGlyAspLeuArgLysLeuLeuAsnLysProGluAsn                               100105110                                                                      CysCysGlyLeuLysGluSerGlnIleLeuSerLeuLeuSerAspIle                               115120125                                                                      GlySerGlyIleArgTyrLeuHisGluAsnLysIleIleHisArgAsp                               130135140                                                                      LeuLysProGluAsnIleValLeuGlnAspValGlyGlyLysIleIle                               145150155160                                                                   HisLysIleIleAspLeuGlyTyrAlaLysAspValAspGlnGlySer                               165170175                                                                      LeuCysThrSerPheValGlyThrLeuGlnTyrLeuAlaProGluLeu                               180185190                                                                      PheGluAsnLysProTyrThrAlaThrValAspTyrTrpSerPheGly                               195200205                                                                      ThrMetValPheGluCysIleAlaGlyTyrArgProPheLeuHisHis                               210215220                                                                      LeuGlnProPheThrTrpHisGluLysIleLysLysLysAspProLys                               225230235240                                                                   CysIlePheAlaCysGluGluMetSerGlyGluValArgPheSerSer                               245250255                                                                      HisLeuProGlnProAsnSerLeuCysSerLeuIleValGluProMet                               260265270                                                                      GluAsnTrpLeuGlnLeuMetLeuAsnTrpAspProGlnGlnArgGly                               275280285                                                                      GlyProValAspLeuThrLeuLysGlnProArgCysPheValLeuMet                               290295300                                                                      AspHisIleLeuAsnLeuLysIleValHisIleLeuAsnMetThrSer                               305310315320                                                                   AlaLysIleIleSerPheLeuLeuProProAspGluSerLeuHisSer                               325330335                                                                      LeuGlnSerArgIleGluArgGluThrGlyIleAsnThrGlySerGln                               340345350                                                                      GluLeuLeuSerGluThrGlyIleSerLeuAspProArgLysProAla                               355360365                                                                      SerGlnCysValLeuAspGlyValArgGlyCysAspSerTyrMetVal                               370375380                                                                      TyrLeuPheAspLysSerLysThrValTyrGluGlyProPheAlaSer                               385390395400                                                                   ArgSerLeuSerAspCysValAsnTyrIleValGlnAspSerLysIle                               405410415                                                                      GlnLeuProIleIleGlnLeuArgLysValTrpAlaGluAlaValHis                               420425430                                                                      TyrValSerGlyLeuLysGluAspTyrSerArgLeuPheGlnGlyGln                               435440445                                                                      ArgAlaAlaMetLeuSerLeuLeuArgTyrAsnAlaAsnLeuThrLys                               450455460                                                                      MetLysAsnThrLeuIleSerAlaSerGlnGlnLeuLysAlaLysLeu                               465470475480                                                                   GluPhePheHisLysSerIleGlnLeuAspLeuGluArgTyrSerGlu                               485490495                                                                      GlnMetThrTyrGlyIleSerSerGluLysMetLeuLysAlaTrpLys                               500505510                                                                      GluMetGluGluLysAlaIleHisTyrAlaGluValGlyValIleGly                               515520525                                                                      TyrLeuGluAspGlnIleMetSerLeuHisAlaGluIleMetGluLeu                               530535540                                                                      GlnLysSerProTyrGlyArgArgGlnGlyAspLeuMetGluSerLeu                               545550555560                                                                   GluGlnArgAlaIleAspLeuTyrLysGlnLeuLysHisArgProSer                               565570575                                                                      AspHisSerTyrSerAspSerThrGluMetValLysIleIleValHis                               580585590                                                                      ThrValGlnSerGlnAspArgValLeuLysGluLeuPheGlyHisLeu                               595600605                                                                      SerLysLeuLeuGlyCysLysGlnLysIleIleAspLeuLeuProLys                               610615620                                                                      ValGluValAlaLeuSerAsnIleLysGluAlaAspAsnThrValMet                               625630635640                                                                   PheMetGlnGlyLysArgGlnLysGluIleTrpHisLeuLeuLysIle                               645650655                                                                      AlaCysThrGlnSerSerAlaArgSerLeuValGlySerSerLeuGlu                               660665670                                                                      GlyAlaValThrProGlnThrSerAlaTrpLeuProProThrSerAla                               675680685                                                                      GluHisAspHisSerLeuSerCysValValThrProGlnAspGlyGlu                               690695700                                                                      ThrSerAlaGlnMetIleGluGluAsnLeuAsnCysLeuGlyHisLeu                               705710715720                                                                   SerThrIleIleHisGluAlaAsnGluGluGlnGlyAsnSerMetMet                               725730735                                                                      AsnLeuAspTrpSerTrpLeuThrGlu                                                    740745                                                                         __________________________________________________________________________ 

What is claimed is:
 1. An isolated or recombinant nucleic acid comprising at least 96 consecutive nucleotides of SEQ ID NO:
 1. 2. A cell comprising the nucleic acid according to claim
 1. 3. An isolated or recombinant nucleic acid according to claim 1, comprising the sequence set forth as SEQ ID NO:
 1. 4. A cell comprising the nucleic acid according to claim
 3. 5. An isolated or recombinant nucleic acid encoding a polypeptide comprising at least 31 consecutive residues of the amino acid sequence set forth as SEQ ID NO:
 2. 6. A cell comprising the nucleic acid according to claim
 5. 7. A method of making an isolated polypeptide, said method comprising the steps of:introducing the recombinant nucleic acid of claim 5 into a host cell or cellular extract, incubating said host cell or cellular extract under conditions whereby said polypeptide is expressed; and isolating said polypeptide.
 8. An isolated or recombinant nucleic acid according to claim 5 encoding a polypeptide comprising the amino acid sequence set forth as SEQ ID NO:
 2. 9. A cell comprising the nucleic acid according to claim
 8. 10. A method of making an isolated polypeptide, said method comprising the steps of:introducing the recombinant nucleic acid of claim 8 into a host cell or cellular extract, incubating said host cell or cellular extract under conditions whereby said polypeptide is expressed; and isolating said polypeptide.
 11. A method of making an isolated polypeptide, said method comprising the steps of:introducing the recombinant nucleic acid encoding a polypeptide comprising at least 11 consecutive residues of the amino acid sequence set forth as SEQ ID NO: 2 into a host cell or cellular extract, incubating said host cell or cellular extract under conditions whereby said polypeptide is expressed; and isolating said polypeptide.
 12. A method of making an isolated polypeptide according to claim 11, said method comprising the steps of:introducing the recombinant nucleic acid encoding a polypeptide comprising at least 21 consecutive residues of the amino acid sequence set forth as SEQ ID NO: 2 into a host cell or cellular extract, incubating said host cell or cellular extract under conditions whereby said polypeptide is expressed; and isolating said polypeptide. 